The present invention relates to a novel glycoprotein which may participate in differentiation of chondrocytes. The new glycoprotein according to this invention may be also applied as a marker for differentiation of chondrocytes.
Animal cartilage tissues are composed of chondrocytes and matrices. Cartilage tissues constitute a major portion of skeleton during the embryonic stage and, after birth, they are replaced by bone through cartilaginous osteogenesis. At the onset of cartilaginous osteogenesis, cartilage tissues would be transformed from quiescent cartilage tissues to proliferative cartilage tissues and successively differentiated to hypertrophic cartilage tissues. As discussed above, it has been well-known that chondrocytes are essential cells particularly for osteogenesis during the growth period. However, there remain many aspects not yet elucidated in regard to differentiation of chondrocytes and cartilaginous osteogenesis.
On the other hand, Yan et al. [Yan et al.; J. Biol. Chem. vol. 265, pages 10125-10131, 1990)]and Kato et al. [Kato et al., Japanese Journal of Bone Metabolism, vol. 10, No. 2, pages 187-192 (1992)]have studied the effect of lectin on differentiation and proliferation of chondrocytes, and, inter alia, elucidated that concanavalin A, which is a jack bean lectin and has an affinity to .alpha.-D-mannose residues and .alpha.-D-glucose residues, can strongly increase synthesis of proteoglycan, which is a marker of differentiation of chondrocytes. There remain, however, many unclear points of its mechanism, wherein what receptor molecules concanavalin A may be bound to on the cell membrane of chondrocytes, how its stimulus may be transferred to such target sites as the nucleus in the cells and so on.
Moreover, it has been reported that chondrocytes, when excess retinoic acid (identical with Vitamin A) is added, may be modified to the so-called dedifferentiated type of cells, wherein they may be deformed in morphology from the spherical cells peculiar to differentiated chondrocytes to the fibroblast-like flat, expanded cellular form with an increased expansibility and may biochemically lose a synthesis ability of glicosaminoglycan (GAG), which is a differentiation marker for chondrocytes and other [Benya et al., Develop. Biol., vol. 118, pages 296-305 (1986)]. However, there has not yet been reported any relationship between the reactivity of chondrocytes to concanavalin A and the differentiation of chondrocytes.
Hence the problem of the present invention was to elucidate the substance capable of controlling the function of chondrocytes and, in the end, the substance capable of controlling enhancement of osteogenesis and to elucidate the substance relating to the differentiation of chondrocytes. This substance leads to a new type of therapy, prophylaxis and diagnosis of cartilaginous metabolic diseases and/or metabolic bone diseases.
In order to solve the above-mentioned problem, the present inventors have attempted to obtain a new membrane glycoprotein on the chondrocyte membrane bound to concanavalin A having a potent differentiation-enhancing activity to chondrocytes. And further, the present inventors have also elucidated a relationship between the protein thus obtained and the differentiation of chondrocytes by comparative tests with dedifferentiated cells having no more properties peculiar to chondrocytes with retinoic acid added.
Hence the present invention refers to a concanavalin A-binding protein (CMP) existing in chondrocytes, but not in dedifferentiated cells from chondrocytes. In a preferred embodiment CMP has a molecular weight of about 76 KD or of about 67 KD after treatment endoglycosidase as analyzed by SDS-PAGE. In a further particularly preferred embodiment CMP contains at least the N-terminal amino acid sequence ##STR1## wherein Xaa means an unknown amino acid.
CMP can be obtained by a method containing the following steps:
(a) isolating chondrocytes; PA1 (b) separating membrane proteins from the chondrocytes of step (a); PA1 (c) isolating concanavalin A-binding glycoproteins through a concanavalin A-affinity column; and PA1 (d) isolating CMP,
preferably by a method wherein prior to step (c) other membrane proteins than concanavalin A-binding glycoproteins were separated.
Most preferred, CMP can be isolated by the following method:
As to the materials for chondrocytes, one may use cartilaginous tissues of various animals and one may obtain chondrocytes, for example, by treatment of the material of costal cartilage growth plates of rabbits with protease and collagenase according to the method by Kato et al. [Kato et al., J. Cell Biol., vol. 100, pages 477-485 (1985,)]. The chondrocytes thus separated can be incubated in a medium containing fetal calf serum (FCS) in petri plates at 37.degree. C. under environment of 5% CO.sub.2 and 95% air. The chondrocytes thus incubated can be recovered, fractioned by means of a homogenizer and subjected e.g. to precipitation equilibrium centrifugation with sucrose equilibrium density-gradients of 17%/40% in order to recover membrane proteins. The membrane proteins thus recovered can be developed directly with a concanavalin A-affinity column or in a preferred embodiment previously developed with an affinity column using, for example, a representative lectin, a wheat germ lectin for removing other membrane proteins bound to lectins than concanavalin A and thereafter developed with a concanavalin A-affinity column, or using other methods to further fractionate concanavalin A-binding protein fractions. Dedifferentiated cartilage cells are derived and formed with various concentrations of retinoic acids and concanavalin A-binding protein fractions are also fractionated. The specificity of the concanavalin A-binding proteins thus obtained to cartilage cells can be investigated by comparing these fractions by way of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-Page analysis of the deglycosilated forms of CMP was also carried out. The glycoprotein specific to the aimed cartilage cells was characterized by amino acid sequencing of the N-terminus.
CMP provided according to this invention is the substance that relates to differentiation of chondrocytes and also to control of functions by chondrocytes and control of cartilaginous osteogenesis. Therefore, CMP or antibodis against CMP can be utilized as a new type of therapy, prophylaxis and diagnosis against cartilaginous metabolic diseases and/or metabolic bone diseases. Therefore, the present invention refers also to a pharmaceutical or a diagnostic aid containing CMP or antibodis against CMP.
The present invention will be explained in detail by way of the following examples, which are not to be construed to limit the present invention.